Synthesis of steroids



United States Patent 3,359,179 SYNTHESIS OF STEROIDS Patrick A. Diassi,Westfield, and Pacifico A. Principe, South River, N.J., assignors, bymesne assignments, to E. R. Squibb & Sons, Inc., New York, N.Y., acorporation of Delaware N 0 Drawing. Original application Mar. 23, 1965,Ser. No. 442,203, now Patent No. 3,316,281, dated Apr. 25, 1967. Dividedand this application Sept. 27, 1966, Ser. No. 612,061

2 Claims. (Cl. 195-51) ABSTRACT OF THE DISCLOSURE Procedure forpreparing 4a,8,14-tr-imethyl 1'8 nor-5a,8a,9fl,14B-androst-1-en-3,11,17-trione and4a,8,'l4-trimethyl-17-hydroxy 18 nor 5a,8ot,9fi,l4fi androstane-3,11-dione utilizing Fusarium' javanicum in the substrate.

Synthesis of steroids This application is a divisional application ofour application, Ser. No. 442,203, filed Mar. 23, 1965, now U.S. PatentNo. 3,316,281.

This invention relates to and has for its object the provision of newphysiologically active compounds, and more particularly, compoundsselected from the group consisting of 40,8,14tIlII16'[llyl-18-H01-5a,8a,9/3, 14,8-androst-1-en 3,1-1,17-trione and4a,8,14-trimethyl-17-hydroxy-18-nor- 5oc,3zx,9,8,l4B-8J1drOStan6-3, 1l-dione.

The novel compounds of this invention are pharmacologically activesubstances and are particularly useful as anti-androgenic,anti-estrogenic and anti-gonadotrophic agents.

The compounds may be formulated for such administration, theconcentration and/or dosage being based on the activity of theparticular compound and the requirements of the patient.

The =final products of this invention are prepared by the process ofthis invention which entails utilizing 4u,8,l4-trimethyl-l-8-nor-5a,8e96,l4fl-androstane-3,l1,17 trione as the startingreactant. This compound may be prepared by refluxing 404,8,14 trimethyl16B hydroxy 18-nor- 5m,8a,9fl,l3a,14fi-androstane-3,11,17-trione16-acetate in the presence of glacial acetic acid and zinc dust.

It has been found that the compounds of this invention can be preparedfrom the starting reactant by subjecting the latter to the action of amicroorganism of the genus Fusarium or to the action of the enzymesthereof under oxidizing and preferably aerobic conditions.

To prepare the compounds of this invention, 4a,8,l4- trimethyl18-nor-5a,8ot,9,8,14 3-androstane-3,11,17-trione may be first subjectedto the action of enzymes of a microorganism of the genus Fu-sarium underoxidizing conditions. This oxidation can best be effected either byincluding the starting reactant in an aerobic culture of the microorganism, or by bringing together in an aqueous medium, the compounds,air, and enzymes of non-proliferating cells of the microorganism.

In general, the conditions of culturing the Fusarium microorganism forthe purposes of this invention are (except for the inclusion of thestarting material to be con verted), the same as those of culturingvarious other microorganisms for the production of antibiotics, vitaminB-12, and other like substances. The microorganism is grown aerobicallyin contact with, (in or on) suitable fermentation medium. A suitablemedium essentially comprises a source of carbon and energy. The lattermay be a carbohydrate, for example, molasses, glucose, maltose,

ice

starch or dextrin, a fatty acid, a fat and/or the compound itself.Preferably however, the medium includes an assimilable source of carbonand energy in addition to the steroid. Among the fats utilizable for thepurpose of this invention are lard oil, soybean oil, linseed oil,cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesameoil, crude palm oil, fancy mutton tallow, sperm oil, olive oil,tristearin, tripalmitin, triolein and trilaurin. Among the fatty acidsutilizable for the purpose of this invention are stearic acid, palmiticacid, oleic acid, linoleic acid and myristic acid.

The source of nitrogenous factors utilizable for the purposes of thisinvention may be organic (e.g., soybean meal, cornsteep liquor, yeastextract, meat extract and/or distillers solubles) or synthetic (i.e.,composes of simple, synthesiza-ble organic or inorganic compounds, suchas ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile air supply should be maintained during fermentation,for example, by the conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated culture. Thecompound may be added to the culture during the incubation period, orincluded in the medium prior to sterilization or inoculation. Thepreferred (but not limiting) range of the concentration of the compoundin the culture is about 0.01% to about 0.1%. The culture period (orrather the time of subjecting the compound to the action of the enzyme)may vary considerably, the range of about twenty-four to ninety-sixhours being feasible, but not limiting.

The microbial process described hereinabove yields the 412,8,14trimethyl l8 nor 5u,8a,9;8,14,8-androst-d-en- 3,11,17-trione andadditionally upon further processing, there is obtained 4a,8,l4-trimethyl-17-hydroxy-18-nor-5a, 8 9/3,14B-androstane-3,-1 l-dione.

The invention may be illustrated by the following examples, alltemperatures are in degrees centigrade unless otherwise stated.

EXAMPLE 1 4a,8,1 4-trimethyl-1 8-n0r-5 a,8oc,9,3,1 4 3-andr0stane-3,11,17-tri0ne EXAMPLE 2 Surface growth from each of 2 two-Week-old agarslants of Fusarium javanicum var. ensiforme (QM-524) (ArmyQuartermaster, Natick, Mass), the slants containing as a nutrient medium(A):

Grams Glucose 10 Yeast extract 2.5 Kai-IP0 1 Agar 2O Distilled water to1 liter.

is suspended in 5 ml. of 0.1% aqueous sodium lauryl sulfated solution.One ml. portions of this suspension are used to inoculate eight 250 ml.Erlenmeyer flasks, each containing 50 ml. of the following sterilizedmedium (B):

Grams Dextrose 1O Cornsteep liquor 6 NH H PO 3 Yeast extract 2.5 CaCO2.5 Distilled water to 1 liter.

After 24 hours incubation at 25 with continuous rotary agitation (280cycles/minute; two inch radius), 10% (v.:v.) transfers are made to forty250 ml. Erlenmeyer flasks, each containing 50 ml. of freshly sterilizedmedium B. After 18 hours of further incubation, using the sameconditions described above, each fiask is supplemented with 300micrograms/ml. of 4a,8,l4-trimethyl-18-nor- 5u,8u,9/3,14Bandrostane3,11,17 trione. The steroid is added by supplementing each flask with0.25 ml. of a sterile solution of the steroid in -N,N-dimethylformamidecontaining 60 mg./m1. of steroid. A total of 600 mg. is fermented. After7 days of further incubation, using the same conditions as describedabove, the contents of the flasks are pooled and filtered through aSeitz clarifying pad. The flasks, mycelium and pad are washed withsuccessive 50 ml. portions of warm water. The combined filtrate andwashings have a volume of 2500 ml. They are extracted three times with800 ml. portions of chloroform which are combined, washed twice with 1liter portions of water and evaporated, in vacuo. The residue is platechromatographed on Woelm neutral alumina (Activity V) using chloroformas the developing solvent. The band detectable by UV at Rf-0.5 is elutedwith ethyl acetate, evaporated and crystallized from acetonehexane togive 92 mg. of 40:,8,14-trimethy1-18-nor-5u,8a,9/3,14B-

androst-l-en-3,11,17-trione having a melting point about 205-207" C.,[041 37 (chloroform),

A 249 Inn (5, 10,270)

Analysis.Calcd for C H O (328.46): C, 76.79; H, 8.59. Found: C, 76.70;H, 8.51.

A band detectable with iodine at Rf 0.8 is eluted with ethyl acetate,evaporated and crystallized from acetonehexane to give 89 mg. of4m,8,14-trimethyl-l7-hydroxy-18- nor-5a,8a,9B,14B-androstane-3,1l-dionehaving a melting point about 213215 C., [M +37 (chloroform).

AnaIysis.Calcd for C H O (332.49): C, 75.86; H, 9.70. Found: C, 75.70;H, 9.84.

While there have been described various embodiments of the invention,the compositions and methods described are not intended to be understoodas limiting the scope of the invention, as it is realized that changestherein are possible and it is further intended that each elementrecited in any of the following claims is to be understood as referringto all equivalent elements for accomplishing sub stantially the sameresults in substantially the same or equivalent manner, it covering theinvention broadly in whatever form its principle may be utilized.

What is claimed is:

1. A process for the preparation of 4a,8,14-trimethyl-18-nor-5u,8ot,9fi,14fi-androst-1-en-3,11,17 trione; which comprisessubjecting 401,8,14 trimethyl 18 nor 5a,8a,9}8,14B-androstane-3,l1,17trione to the action of a microorganism ofthe species Fusarium javanicum.

2. A process for the preparation of 4a,8,14-trimethyl-17-hydroxy-18-nor-5a,8:x,9/3,14B-androstane 3,11 dione which comprisessubjecting 4a,8,14 trimethyl 18 nor-5a,8a,9;8,14,8-androstane-3,-11,17-trione to the action of amicroorganism of the species Fusarium javanicum.

No references cited.

A. E. TANE'NHOLTZ, Primary Examiner.

1. A PROCESS FOR TH PREPARATION OF4A,8,14-TRIMETHYL18-NOR-5A,8A,9B,14B-ANDROST-1-EN-3,11,17 - TRIONE;WHICH COMPRISES SUBJECTING 4A,8,14 - TRIMETHYL - 18 - NOR - 5A,8A,9B,14B-ANDROSTANE-3,11,17-TRIONE TO THE ACTION OF A MICROORGANISM OFTHE SPECIES FUSARIUM JAVANICUM.
 2. A PROCESS FOR THE PREPARTION OF4A,8,14-TRIMETHYL17-HYDROXY-18-NOR-5A,8A,9B,14B-ANDROSTANE - 3,11 -DIONE WHICH COMPRISES SUBJECTING 4A,8,14 - TRIMETHYL - 18 -NOR5A,8A,9B,14B-ANDROSTANE-3,11,17-TRIONE TO THE ACTION OF AMICROORGANISM OF THE SPECIES FUSARIUM JAVANICUM.